Methods:
Rat retinal endothelial cells (RRECs) were grown in normal (5 mM glucose; N) or high glucose (30 mM; HG) medium for 7 days in either sparse or dense conditions. In parallel, cells grown in N or HG medium were exposed to a connexin channel blocker 18-β-glycyrrhetinic acid (GA) or or a non-active analogue of GA, glycyrrhizic acid (GZ) as control. To determine hemichannel activity, the dye uptake method was employed. Cells undergoing apoptosis was assessed using differential staining with acridine orange/ethidium bromide and TUNEL assay.

Results:
Dye uptake results indicate RRECs grown in sparse condition exhibit increased hemichannel activity compared to those grown in densely populated cell condition. Interestingly, while HG tends to increase hemichannel activity in cells grown under sparsely populated condition, HG appeared to reduce hemichannel activity in densely populated cell condition. Importantly, when hemichannel activity was blocked apoptosis was observed, and at increasing concentrations of GA apoptosis was accelerated, whereas GZ had no effect. Thus, a biphasic effect of HG was observed as cells proliferated from a sparse to a dense, compact condition: an initial upregulation of hemichannel activity followed by sustained uncoupling effect.

Conclusions:
Findings from this study indicate that Cx43 hemichannels play a distinctive role in response to HG in retinal endothelial cells in contrast to its role in gap junction intercellular communication activity. Importantly, increased hemichannel activity in the sparse cell populations under HG condition may signal a survival response countering HG-induced apoptosis in the pathogenesis of diabetic retinopathy.